The elevation of growth hormone (GH) levels in animals, e.g., mammals including humans, upon administration of GH-releasing compounds can lead to enhanced body weight. Ghrelin, identified as an endogenous ligand for the GH secretagogue receptor (GHS-R) is a powerful stimulator of pulsatile GH secretion and exhibits intricate interactions with the primary hypothalamic GH regulators (1-3). Ghrelin as well as growth hormone releasing peptides (GHRPs) and growth hormone secretagogues (GHSs) also function as potent orexigenic peptides (4-7). Initial peptide antagonists that inhibited the binding activity of GHSs in hypothalamic tissue in vitro were reported in 1991-92 (8-10). This included the Substance P analog, [Arg1 DPhe5 DTrp7,9 Leu11]-Substance P, that subsequently was demonstrated by Hoist et al (vide infra) to have both inverse agonist and ghrelin-R antagonist activity. Orexigenic compounds stimulate appetite.
Ghrelin is a 28 amino acid peptide, which has a unique structure among peptide hormones as it is acylated at Ser3 usually with an n-octanyl moiety (Bednarek et al., 2000; Kojima et al., 1999). This post-translational modification is essential for the activity of the hormone—as mediated through the seven transmembrane 0 (7TMG) protein coupled ghrelin receptor—both in vitro and in vivo (Kojima et al., 1999; Nakazato et al., 2001; Tschop et al., 2000).
Plasma levels of ghrelin rise precipitously in the blood before meals, when the stomach is empty, and fall after or during food consumption. Since intracardiac venous (i.v.) or intracerebroventricular (i.c.v) administration of ghrelin increases food intake, it appears that the physiological role of ghrelin is a link or messenger between the stomach and the hypothalamus and the pituitary. One hypothesis is that when an organism is getting ready for a meal, the CNS sends signals to the GI tract telling that a meal is about to be consumed in order to obtain information back about the status of the digestive process, state of distension etc. from the various chemical and mechanical sensors in the gut. Here, ghrelin could be an important hormonal messenger, which is sent back to the central nervous system (CNS) as a signal telling that there is no food in the stomach and that the gastrointestinal (GI) tract is ready for a new meal. In such a paradigm it is clear that a blocker of the ghrelin receptor would be a very efficient anti-obesity agent, as it would block the meal initiating, appetite signal from the GI tract.
The ghrelin receptor, GHS-R1a, belongs to a relatively small family of 7 transmembrane G-protein coupled receptors (11). A number of findings demonstrate how the ghrelin receptor may uniquely play a role in mediating the action on GH release and food intake. This includes ghrelin receptor genetics, mutations, structure, intracellular signaling, high constitutive activity, enhancement of the number of hypothalamic ghrelin receptors during starvation, etc. A spectrum of growth and metabolic changes occur in mice as a result of knockout of the ghrelin molecule as well as the ghrelin receptor. Adiposity in mice followed overexpression of the ghrelin receptor in hypothalamic growth hormone releasing hormone (GHRH) arcuate neurons. Over time, select biological effects of ghrelin/GHSs, especially non-endocrine effects, have been revealed which presumably occur via subtypes receptors of ghrelin or perhaps ghrelin receptors with select mutations. Evidence indicates binding and activation of the multifunctional CD36 receptor by GHSs. Another noteworthy finding of the ghrelin receptor was that under pathophysiological conditions the density of this receptor was reported to be five times greater in atherosclerotic coronary arteries (12).
Holst and Schwartz characterized the high constitutive activity of this receptor. Also, they demonstrated inhibition of the constitutive activity of the [DArg1, DPhe5, DTrp7,9 Leu11]-substance P analog which has been previously characterized both in vitro and in vivo as a weak competitive receptor antagonist to acute and chronic actions of GHRP-2 and ghrelin. These investigators demonstrated in vitro that this analog has 2 types of ghrelin receptor inhibiting activities. At a low dose (5 nM, IC50), this Sub P analog is a potent inverse receptor agonist since it decreases elevated intracellular IP3 levels in the absence of ghrelin but also it is a weak ghrelin GHRP-6 competitive receptor antagonist since high dosages (630 nM, IC50) inhibit receptor binding of both peptides (13, 14). Petersen, Holst, Schwartz et. al. reported that continuous i.c.v 7 day infusion of a very low dose of the Sub P ghrelin receptor inverse agonist inhibited body weight gain of male rats (15). This was a dose that would be too low to function as a competitive ghrelin receptor antagonist and thus it was considered to be due to the inverse agonist activity of the Sub P analog. In vitro evidence supports GHS-R antagonists with only inverse agonist or only ghrelin/GHS-R activity or a combination of the two (16).
Another possible novel functional role of the high constitutive activity of the ghrelin receptor in the CNS was proposed by Zigman et al on the distribution and functional implication of the ghrelin receptor in the brain of the rat and mouse (17). They proposed that the high constitutive activity of the ghrelin receptor plays a key functional role at CNS sites at which the receptor is expressed within the blood brain barrier and thus does not have immediate access to circulating ghrelin. This is in contrast to the ghrelin receptor located in the arcuate nucleus and dorsal vagal complex role. Thus it is possible that select GHSs, because of their different chemistry, may have ready access to brain sites inaccessible to ghrelin. If this occurs, GHSs' actions at these sites may alter the CNS ghrelin constitutive activity via receptor number and/or activity.
Although regulation of food intake by numerous hormones reveals its complexity, the inhibition of ghrelin induced food intake implies a fundamental biological functional aspect of the ghrelin system. For example, in the absence of the ghrelin receptor, transgenic female and male mice fed a high fat diet eat less food, less of the consumed calories are stored, fat is more of the energy substrate, and body weight and body fat are less in these mice than control mice (17, 18). When the ghrelin receptor was absent and mice were fed a normal diet, body weight and body fat were decreased in female but not in male mice. In the absence of the ghrelin peptide, transgenic male mice (female mice not studied) had less rapid body weight gain on a high fat diet (19). This was associated with increased energy expenditure and increased locomotive activity as well as decreased adiposity. Both of these studies indicate the ghrelin system is involved in body weight control especially when consuming a high caloric type of obese inducing diet. In the absence of the ghrelin receptor (GHS-R1a), ghrelin no longer increased food intake. Thus, the singularity of this receptor for mediating ghrelin induced food intake is indicated. Also, hyperphagia is an established risk factor in diabetes mellitus in humans and evidence indicates that sub-threshold doses of ghrelin increases food intake in streptozotocin treated rats (20). Experimental studies in rats revealed interrelationships of ghrelin, somatostatin and GHRH on function of the GH axis (21, 22).
Thus, compounds which effectively inhibit the ghrelin receptor are needed to disrupt the activity of ghrelin at the level of the CNS. Such compounds would be useful in the treatment of metabolic diseases and disorders such as obesity, diabetes mellitus, and inhibition of growth hormone secreted from tumors such as pituitary, prostate, osteoblast, pancreatic and hepatoma. Relevant basic and clinical ghrelin and GHS data have been discussed by Bowers et al (23, 24) as well as by other investigators (25-27).